Bertrand-Burggraf E, Lefèvre J F, Daune M
Nucleic Acids Res. 1984 Feb 10;12(3):1697-706. doi: 10.1093/nar/12.3.1697.
In order to follow the kinetics of the initiation of transcription by the E. coli RNA polymerase, we have used the procedure of abortive initiation as described by Mc Clure (1980) (7). In place of radioactive labeling we have taken advantage of a fluorescent probe (UTP gamma ANS) to obtain fast and accurate determinations of the rate of transcription and to deduce from kinetic equations both the binding constant (KB) and the rate of isomerization (k2) which characterize the classical two-step model. This analysis was applied to the tet promoter of pBR322 in a linearized plasmid DNA and was studied in function of temperature (from 25 degrees C to 37 degrees C) and of pH (from 6 to 8.3). The association is entropy driven (delta H degrees = 29 Kcal/mole and delta S degrees = 130 e.u.). The activation energy of isomerization is 13 Kcal/mole. Both k2 and k-2 are increasing with pH. The insensitivity to pH of the KBK2 product could be tentatively explained in terms of the processive aspect of the polymerase binding to its specific site.
为了追踪大肠杆菌RNA聚合酶起始转录的动力学过程，我们采用了麦克卢尔（1980年）（7）所描述的流产起始程序。我们利用荧光探针（UTPγANS）代替放射性标记，以快速准确地测定转录速率，并从动力学方程中推导出表征经典两步模型的结合常数（KB）和异构化速率（k2）。该分析应用于线性化质粒DNA中的pBR322 tet启动子，并研究了温度（从25℃到37℃）和pH值（从6到8.3）的影响。这种结合是由熵驱动的（ΔH° = 29千卡/摩尔，ΔS° = 130熵单位）。异构化的活化能为13千卡/摩尔。k2和k-2都随pH值增加。KBK2产物对pH值不敏感，可以根据聚合酶与其特定位点结合的持续性方面进行初步解释。
